BACK TO WEB VERSION

WINTER 2007
In Search of Crypto's Achilles Heel
by Renee Twombly

One way to get rid of a parasite—an organism that lives on or in a different species—is to destroy the host, as in killing mosquitoes to eliminate the malaria parasite within it. This approach is not desirable when the host is us.

But in the current era of genomic medicine, we are gaining new, elegant, and, as researchers like to say, “rational” ways of dealing with parasitic disease. Such methods, based on knowledge of the structure and functioning of the parasitic organism at the molecular level, involve figuring out its vulnerabilities and designing drugs to target them directly.

At the University of Georgia, researchers in the Center for Tropical and Emerging Global Diseases (CTEGD) are employing that rationale to understand a number of different parasites, some of which cause disease and death worldwide. And one that is piquing considerable interest is Cryptosporidium, which gives people long and uncomfortable bouts of diarrhea, sometimes even killing particularly vulnerable individuals.

Cryptosporidium—or “Crypto” to those who study it—is as tough as any organism known. In its dormant state, as a little hard-shelled spore, it could be stored in sulfuric acid. On a more practical level, the chlorine used to treat drinking water has no effect on the protozoan, thereby putting much of the U.S. water supply at risk for a Crypto outbreak. In fact, more than 400,000 residents of Milwaukee were sickened by Cryptosporidium in their drinking water, and thousands of children in a New York state park became ill when spores rained down on them while playing in a “sprayground.” (See Notable Outbreaks sidebar.)

Not surprisingly, up to 80 percent of the United States population has been exposed to Crypto, according to the Big Bad Bug Book of the U.S. Food and Drug Administration. This conclusion is based on survey evidence that many individuals’ immune systems have produced antibodies to the parasite, meaning they have successfully fended off an infection. Likely, the human hosts never knew what that little gastrointestinal problem they had was all about.

The parasite can also hitch a ride on any food touched by a contaminated food handler or that has been irrigated with contaminated water; small outbreaks have occurred at social events where meals were served. As little as one organism can initiate an infection, the FDA warns.

Crypto belongs to the Phylum Apicomplexa, a large group of single-celled, spore-producing protozoa that includes the parasites responsible for malaria, toxo-plasmosis and an important parasite of chickens, Eimeria.

In its various forms, Cryptosporidium can infect everything from birds to mice—and also chickens, which are important to Georgia’s economy—but it seems to be prevalent among herd animals such as cows, goats, sheep, deer and elk. In the past, when a human got an infection it was thought to come from Cryptosporidium parvum, which targets cows. But it was only because of the Milwaukee event—the nation’s largest outbreak of severe diarrhea—that scientists discovered the Cryptosporidium hominis variant, which can spread from human to human. (See “Milwaukee” Sidebar.)

When a Crypto spore infects a human and travels to the gut, four small cells shoot out from the shell to invade cells in the intestinal lining. Then, like viruses, the parasites multiply within cells, ultimately to infect neighboring cells. Unlike viruses, which are just little strings of DNA or RNA, Crypto are cellular beings living within other cells. “They are really dependent on many contributions by the host cell,” said Boris Striepen, a cellular biologist at CTEGD.

When a Crypto spore infects a cell, it creates a vacuole around itself with a bubble of membrane. It then establishes channel-like pores through this membrane to gain access to the host cell. Critical to this process are membrane-surface “transporters” that allow the parasite to essentially grab anything of interest passing by in the host cell’s fluid. For example, because it doesn’t make sugar as a fuel, Crypto has a sugar transporter.

“All the ingredients needed for cellular metabolism are pretty freely available in the cytoplasm of the host cell,” Striepen said. “So they take it out of your cell and bring it into theirs. They are using the host cell for food and shelter.”

The organism is really well put together for survival purposes, he added. “They are set up to succeed, and they succeed fairly often.”

Striepen works with CTEGD colleague Jessica Kissinger, an expert in parasite genomics, to help figure out which genes in Crypto are doing what. With collaborators at the University of Pennsylvania, Kissinger is funded to make a database of all known apicomplexan genes, and this includes “CryptoDB”—a database containing the estimated 4,000 genes present in each of the two Crypto species. This database allows Kissinger to do “data mining,” which she describes as a systematic way of “looking for a needle in a haystack.”

Striepen will run experiments based on a genetics question that Kissinger is trying to answer, and Kissinger will search in her database for a gene that pops up in one of Striepen’s experiments. “The experimental and the computational approaches each have their own strengths and weaknesses, but combining them is very powerful,” Striepen said.

Parallel to Striepen and Kissinger’s interest in understanding basic parasitic biology is their hunt for potential drug targets—agents that can be used to treat individuals with Crypto infections. The scientists’ strategy is to identify areas in which the biology of the parasite is radically different from that of the host.

“Because Cryptosporidium sits on the same branch of the tree of life as we do, this makes an infection hard to treat,” said Striepen. “We have really good drugs against bacteria, which are completely different from humans, but we don’t have as good, as specific, or as effective drugs for pathogens that are more similar to ourselves.”

Striepen and Kissinger decided that the most straightforward avenue for finding exploitable differences is metabolic activity—the modification of chemical compounds within a cell. “For example, if we make a certain amino acid and parasites make it using completely different enzymatic machinery, then we could theoretically hit that system without fearing that we’d interfere with our own metabolism, which could cause side effects,” Striepen said.

This strategy appears to be succeeding. Striepen and Kissinger’s teamwork has already led to one potential drug target, and a novel discovery. They found that Crypto is a genetic mix of species—a lot of protozoan and a little bit of bacteria, with apparently something borrowed from the plant world as well.

Unlike the typical mode of “vertical” genetic transmission, in which the genes of an infant are derived from its two parents, nature sometimes exhibits “horizontal” gene transfer—genes that jump from one living organism to another. This process is often seen in bacteria, which love to share genes that help them survive, Striepen said. “That is why there is so much antibiotic-drug resistance today and why it is spreading.”

Striepen and Kissinger published a series of studies that made the case this process happens in Cryptosporidium parvum as well.

Based on Striepen’s lab work that examined activity of the protozoan and Kissinger’s analysis of the Crypto genome, they found that Cryptosporidium parvum carries bacterial genes and that these genes are needed to help it make DNA. Their finding may help them locate biological “back doors” into Crypto—enzymes and proteins, made by bacterial genes, that can be targeted by novel drugs that will not affect human cells.

Striepen believes he has already found such a potential Crypto Achilles heel. Working with scientist Lizbeth Hedstrom from Brandeis University, he tested 45,000 different chemicals for the ability to inhibit the bacterial enzyme that helps assemble the parasite’s DNA. So far, they have identified 10 good candidates—compounds that can block the enzyme.

The transporters that the parasite uses to bring in material it needs from the host cell might also make a good drug target, Kissinger said, so she and her research team are assembling a diagram of metabolic activity associated with the parasitic genome, a process they dub “metabolic-profiling.”

“If we can figure out which transporters are bringing in sugars, which ones bring in amino acids, how this thing eats and makes the compounds it needs, that could give us a variety of ways to disrupt their activity,” she said.

Striepen works with CTEGD colleague Jessica Kissinger, an expert in parasite genomics, to help figure out which genes in Crypto are doing what. With collaborators at the University of Pennsylvania, Kissinger is funded to make a database of all known apicomplexan genes, and this includes “CryptoDB”—a database containing the estimated 4,000 genes present in each of the two Crypto species. This database allows Kissinger to do “data mining,” which she describes as a systematic way of “looking for a needle in a haystack.”

Striepen will run experiments based on a genetics question that Kissinger is trying to answer, and Kissinger will search in her database for a gene that pops up in one of Striepen’s experiments. “The experimental and the computational approaches each have their own strengths and weaknesses, but combining them is very powerful,” Striepen said.

Parallel to Striepen and Kissinger’s interest in understanding basic parasitic biology is their hunt for potential drug targets—agents that can be used to treat individuals with Crypto infections. The scientists’ strategy is to identify areas in which the biology of the parasite is radically different from that of the host.

“Because Cryptosporidium sits on the same branch of the tree of life as we do, this makes an infection hard to treat,” said Striepen. “We have really good drugs against bacteria, which are completely different from humans, but we don’t have as good, as specific, or as effective drugs for pathogens that are more similar to ourselves.”

Striepen and Kissinger decided that the most straightforward avenue for finding exploitable differences is metabolic activity—the modification of chemical compounds within a cell. “For example, if we make a certain amino acid and parasites make it using completely different enzymatic machinery, then we could theoretically hit that system without fearing that we’d interfere with our own metabolism, which could cause side effects,” Striepen said.

This strategy appears to be succeeding. Striepen and Kissinger’s teamwork has already led to one potential drug target, and a novel discovery. They found that Crypto is a genetic mix of species—a lot of protozoan and a little bit of bacteria, with apparently something borrowed from the plant world as well.

Unlike the typical mode of “vertical” genetic transmission, in which the genes of an infant are derived from its two parents, nature sometimes exhibits “horizontal” gene transfer—genes that jump from one living organism to another. This process is often seen in bacteria, which love to share genes that help them survive, Striepen said. “That is why there is so much antibiotic-drug resistance today and why it is spreading.”

Striepen and Kissinger published a series of studies that made the case this process happens in Cryptosporidium parvum as well.

Based on Striepen’s lab work that examined activity of the protozoan and Kissinger’s analysis of the Crypto genome, they found that Cryptosporidium parvum carries bacterial genes and that these genes are needed to help it make DNA. Their finding may help them locate biological “back doors” into Crypto—enzymes and proteins, made by bacterial genes, that can be targeted by novel drugs that will not affect human cells.

Striepen believes he has already found such a potential Crypto Achilles heel. Working with scientist Lizbeth Hedstrom from Brandeis University, he tested 45,000 different chemicals for the ability to inhibit the bacterial enzyme that helps assemble the parasite’s DNA. So far, they have identified 10 good candidates—compounds that can block the enzyme.

The transporters that the parasite uses to bring in material it needs from the host cell might also make a good drug target, Kissinger said, so she and her research team are assembling a diagram of metabolic activity associated with the parasitic genome, a process they dub “metabolic-profiling.”

“If we can figure out which transporters are bringing in sugars, which ones bring in amino acids, how this thing eats and makes the compounds it needs, that could give us a variety of ways to disrupt their activity,” she said.

For more information contact Boris Striepen at striepen@cb.uga.edu, Jessica Kissinger at jkissing@uga.edu or Dan Colley at dcolley@uga.edu.